High expression of tight junction protein 1 as a predictive biomarker for bladder cancer grade and staging

Tight junction proteins 1–3 (TJP1–3) are components of tight junctions that can link transmembrane proteins to the actin cytoskeleton, and their incidence directly correlates to metastasis. However, the role of the TJP family in bladder cancer has not been adequately evaluated. In this study, we evaluated the genetic changes, mRNA and protein expressions of the target genes of the TJP family in bladder cancer patients using online database and immunohistochemistry, respectively. We found that TJP1 was amplified in bladder cancer tissue and that the protein expression levels were significantly associated with age (p = 0.03), grade (p = 0.007), and stage (p = 0.011). We also examined the correlation between TJP1 and other high-frequency mutation genes using TIMER. TJP1 mRNA levels were positively correlated with TTN and RYR3 mRNA levels in bladder cancer tissue. Taken together, TJP1 expression is associated with poor clinical outcomes in patients with bladder cancer and can be a useful predictive biomarker for bladder cancer staging.

www.nature.com/scientificreports/ the upregulation of ZO-1 contributes to the invasion and adhesion of melanoma cells 15 . Accumulating evidence suggests that ZO proteins play a central role in cancer progression. However, the role of ZO proteins in bladder cancer has not been elucidated.
In this study, we investigated whether the ZO family (TJP ZO1, ZO-2, and ZO-3, encoded by the TJP1, TJP2, and TJP3 genes, respectively) is associated with the malignant phenotype in bladder cancer. We examined the DNA copy number and mRNA expression of the ZO family in multiple cancer cell lines and cancer patients using online datasets. Based on the analysis of the bioinformation, we identified the target genes and then provided the clinicopathological data for verification relationships in patients with bladder cancer.

Results
In silico mRNA and DNA profiles of TJP family members in multiple cancer cell lines. By evaluating the mRNA expression of TJP1, TJP2, and TJP3 in 40 different cancer cell lines, we found that TJP1, TJP2, and TJP3 were upregulated in 29, 40, and 12 different cancer cell types, respectively (Fig. 1). Next, by examining the DNA copy numbers of the TJP family in multiple cancer cell lines, we observed that the DNA copy numbers of TJP1, TJP2, and TJP3 were upregulated in 10, 9, and 7 different cancer cell lines, respectively (Fig. 2). However, the DNA copy numbers and mRNA expression levels were not consistent.
In silico genetic alterations of TJP family members in bladder cancer patients. The genetic alterations of the TJP family in bladder cancer, examined using cBioPortal, showed that the genetically altered ratios of TJP1, TJP2, and TJP3 were 3%, 3%, and 1.7%, respectively (Fig. 3A). TJP1 and TJP3 had a 2.5-4% amplification ratio and TJP2 has a 5% mutation ratio in bladder cancer patients (Fig. 3B-D). While analyzing the frequency of the co-occurrence of genetically altered TJP1-3 in the same specimen, it was found that genetic alterations of the other TJP family members were not significant in bladder cancer patients (Supplementary Table 1). TJP1 genetic amplification was correlated with mRNA expression in patients with bladder cancer (Supplementary Fig. 1A). In addition, we also compared the TJP1 expression between the protein level and the RNA level from the CCLE dataset (https:// depmap. org/ portal/). Our results found a strong positive correlation in the bladder cancer cell panel (Spearman nonparametric correlation test; correlation coefficient = 0.582; p = 0.05, n = 11) ( Supplementary Fig. 1B). Moreover, we further evaluated expression of TJP1 protein in bladder cancer cell lines. The results showed TJP1 protein was upregulated in bladder cancer cells ( Supplementary Fig. 1C). A previous study showed that TJP1 expression is correlated with cell motility in bladder cancer cells 16 . Therefore, in this study, we focused on TJP1 for further investigation of bladder cancer. TJP1 protein up-regulation is associated with poor clinical outcomes in bladder cancer. As shown in Fig. 4A, TJP1 expression in cancer tissues was classified into two groups (low and high) base on cutoff point, which was set at the median. It was found that high TJP1 expression levels in bladder cancer tissues were significantly associated with age (p = 0.03), grade (p = 0.007), and stage (p = 0.011) ( Table 1). IHC staining results show that TJP1 was significantly upregulated in bladder cancer specimens compared to normal bladder tissues (Fig. 4B). The results also showed that TJP1 was significantly upregulated in the urothelial carcinoma group in bladder cancer specimens compared to normal bladder tissues (Fig. 4C).

TJP1 expression positive correlates with TTN in bladder cancer patients. It was observed that
TTN, RYR3, TRPM1, RB1, ULK4P3, CHRFAM7A, FAN1, and HERC2 were significantly altered in patients with bladder cancer (Fig. 5A). Furthermore, Fig. 5B shows that TJP1 genetically altered co-occurrence in a series of core genes, including TTN, TP53, and RYR3. We further examined the correlation between the mRNA expression of TJP1 and TTN, TP53, and RYR3 by TIMER 17 . The results showed that TJP1 mRNA levels were positively correlated with TTN and RYR3 mRNA levels in bladder cancer tissues (Fig. 5C). Moreover, we examined the protein-protein interactions network by using STRING dataset 18 . The results showed TJP1 interaction with TTN and RYR3 via TP53 ( Fig. 5D and Supplementary Table 2).

TJP family expression correlated with chemotherapy response in bladder cancer cells. It has
been shown that genetic determinants for chemotherapy and radiotherapy response in bladder cancer 19 . To examine the correlation between TJP family mRNA expression and chemotherapy drugs in bladder cancer cells. We found TJP2 expression was positively correlated with IC50 of cisplatin, and TJP3 expression was positively correlated with IC50 of mitomycin C in bladder cancer cell lines (

Discussion
TJP1, also known as zona occludens 1 (ZO-1), is a tight junction protein that can regulate actin cytoskeleton remodeling 20 . Altered expression of TJP1 is found in many cancers and is responsible for modulating cancer migration and invasion 15,[20][21][22] . In this study, the TJP family was evaluated in multiple cancer cell lines and it is a predictive biomarker for bladder cancer staging.
Our data showed that TJP1 is upregulated in multiple cancer cell lines (Fig. 1A). TJP expression regulates several signaling and transcriptional pathways in cancer 23 and is involved in the epithelial-mesenchymal transition (EMT) associated with tumor invasion 24 . Downregulation of TJP1 expression has been observed in gastrointestinal adenocarcinoma, breast cancer, and colorectal carcinoma [25][26][27] . TJP1 expression is regulated by E-cadherin in breast carcinoma 26 . During EMT, downregulation of E-cadherin is accompanied by the upregulation of N-cadherin expression, which promotes cell motility and survival advantage in the early stage tumor 28,29  www.nature.com/scientificreports/ invasion abilities in the melanoma cell 15 . Specifically, high TJP1 mRNA expression has been reported in patients with bladder cancer. Knockdown of TJP1 inhibits cell proliferation, migration, and invasion in bladder cancer cell lines, while, TJP1 mRNA expression is associated with lymph node metastasis in bladder cancer patients 16 .
Additionally, deletions and mutations of TJP1 promote cancer cell proliferation 25 . In our data showed expression of TJP1 protein was associated with grade and stage in patients with bladder cancer (Table 1). Previous studies have shown that tumor mutational burden is a biomarker for predicting responsiveness to immune checkpoint blockade immunotherapy in several cancer types 30,31 . In our study, we found that TNT, TP53, and RYR3 mutations co-occurred with altered TJP1 in bladder cancer patients. In addition, TJP1 mRNA expression levels were positively correlated with TNT and RYR3 mRNA expression levels in patients with bladder cancer. We could not exclude the possibility that TJP1 amplification or expression is correlated with the response rate to immune checkpoint blockade; however, this is the first study to evaluate the TJP1 genetic alterations in bladder cancer patients. Accumulating evidence has shown genetic altered and expression correlated with chemotherapy response in bladder cancer 19 . We also found TJP2 and TJP3 mRNA expression positively correlated with chemoresistance in bladder cancer cell lines (Table 2). However, TJP1 mRNA expression was not significantly correlated with chemotherapy in bladder cancer cell lines. Further mechanisms are needed to investigate the underlying TJP1 expression and subsequently increased chemosensitivity in bladder cancer.
In conclusion, this study evaluated genetic variations in the TJP1 family by the amplification of TJP family members in bladder cancer patients. TJP1 protein expression correlated to tumor grade and stage, indicating that TJP1 can be used as an independent biomarker for bladder cancer staging.

Materials and methods
In silico genetic and mRNA profiles of the TJP1 family in multiple cancer cell lines and cancer patients. The mRNA expression levels and DNA copy numbers of TJP1, TJP2, and TJP3 in 40 different cancer cell lines were evaluated using the Cell Line Encyclopedia (CCLE) dataset. We stratified cancer cell types into upregulated (median > 0) and downregulated (median < 0). Genetic variations in the TJP1 family and genetic altered ranking in bladder cancer patients were evaluated using the online dataset (cBioPortal, v.3.6.20).     License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.